(1) Field of the Invention
The present application generally relates to antibody detection of metabolites. More specifically, methods and compositions are provided for preparing and using antibodies to detect and quantify 24S-hydroxycholesterol.
(2) Description of the Related Art
24S-hydroxycholesterol (24HC) (structure below) is one of several mono-oxygenated metabolites of cholesterol.

Excess cholesterol in the brain is converted to 24HC by cholesterol 24-hydroxylase (CYP46). Owing to its ability to cross the blood-brain barrier, 24HC is present in peripheral circulation. Because CYP46 is predominantly expressed in the brain, 24HC in plasma originates almost exclusively from the brain (Lütjohann et al., 1996). Interestingly, certain CYP46 single nucleotide polymorphisms are associated with Alzheimer's disease (“AD”)(Garcia et al., 2009). Plasma levels of 24HC correlate with brain and hippocampal size (Bretillon et al., 2000; Koschack et al., 2009). 24HC in plasma or cerebrospinal fluid is an indicator of brain cholesterol turnover, particularly from myelin, and demyelination caused by neuronal degeneration results in an increased flux of 24HC across the blood-brain barrier and into plasma. Thus, active neuronal cells are the major source of 24HC in human circulation and the concentration of 24HC in plasma is considered a surrogate marker for brain cholesterol homeostasis. Additionally, 24HC is a signaling molecule in the brain, inducing ApoE-mediated cholesterol efflux from astrocytes by a direct effect on ApoE transcription, protein synthesis and secretion (Leoni et al., 2010). Direct toxic and inflammatory gene expression-inducing effects of 24HC may also be involved in the pathology of AD or other cognitive dysfunctions (Alexandrov et al., 2005; Kölsch et al., 2004; Zhao et al., 2009). Further, neurodegeneration and the resulting loss of neurons has been found in some studies to result in the reduction of 24HC in plasma in AD, Huntington's disease, and multiple sclerosis (Björkhem et al., 2009; Irizarry, 2004; Leoni, 2009; Leoni et al., 2008; Masterman et al., 2002; Solomon et al., 2009; Teunissen et al., 2003, 2005; Valenza et al., 2007). However, other studies have found increased 24HC plasma levels in AD patients (Lütjohann and von Bergmann, 2003; Lütjohann et al., 2000). In cerebrospinal fluid, 24S-hydroxycholesterol levels increase, and appear to reflect the rate of neurodegeneration (Leoni et al., 2010; Papassotiropoulos et al., 2002; Shafaati et al., 2007). Reduced plasma levels of 24HC are also induced by statin treatment (Lütjohann and von Bergmann, 2003; Vega and Weiner, 2007). In a further association with neurodegenerative disease, gene therapy with CYP46 in a mouse model of AD, administered before the onset of amyloid deposits, reduced Aβ peptides, amyloid deposits and trimeric oligomers, and improved spatial memory in those mice (Hudry et al., 2010). Additionally, ablation of acyl-CoA:cholesterol acetyltransferase 1 (ACAT 1) in the brain leads to a reduction of AD-associated forms of amyloid precursor protein and increases in brain 24HC (Bryleva et al., 2010).
There is a close relationship between the biotransformation of drugs and normal biochemical processes occurring in the human body. The metabolism of drugs involves many pathways associated with the synthesis of endogenous substrates such as steroid hormones, cholesterol and bile acids. It should be recognized that many of the enzymes involved in drug metabolism are principally designed for the metabolism of these endogenous compounds. These enzymes metabolize drugs only because the drugs resemble the natural substrate.
During phase II metabolism, a substrate is rendered more hydrophilic through the covalent attachment of an endogenous molecule. The cytosolic sulfotransferase (SULT) and UDP-glucuronosyltransferase (UGT) families of enzymes account for the majority of phase II metabolism in humans and animals. Sulfonation and glucuronidation are generally considered competing pathways. Typically, sulfonation predominates at low substrate concentrations, while glucuronidation predominates at higher concentrations.
In both brain and liver, 24HC serves as a liver-X receptor (LXR) agonist and has an important role in cholesterol homeostasis. Conversion of this metabolite to bile acids accounts for only 40-50% of the metabolite's elimination, leaving a large percentage of 24HC metabolism and excretion occurring by other pathways. The SULT and UGT enzymes represent a highly responsive defense system against the mutagenicity of carcinogenic environmental chemicals and the toxicity of xenobiotics and endogenous metabolic intermediates. Conjugation with either sulfonate or glucuronic acid has been implicated as important for biliary excretion of 24HC.
Cytosolic SULTs are involved with the conjugation of therapeutic drugs, xenobiotics and small endogenous compounds including hydroxysteroids, thyroid hormones, estrogens, bile acids, cholesterol and oxysterols. 24HC can be conjugated by at least three isoforms of human cytosolic SULTs, but others (e.g. SULTs 1A1, 1A3, 1B1, 1C1) display no discernable activity (Cook et al., 2009). SULTs 2A1 and 1E1 sulfonate both the 3- and 24-hydroxyl groups to form 24-hydroxycholesterol-3,24-disulfate. SULT2B1b forms only 24-hydroxycholesterol-3-sulfate. The 3-sulfate as a monosulfate or as the disulfate can be hydrolyzed by human placental steroid sulfatase, whereas the 24-sulfate is resistant to its actions. Both the 24-hydroxycholesterol-3-sulfate and 24-sulfate are antagonists of LXR activation.
Current methods for determination of 24HC generally involve gas chromatography-mass spectrometry, or liquid chromatography-tandem mass spectrometry (see, e.g., Berkard et al., 2004; DeBarber et al., 2008; Leoni et al., 2008). Because the importance of 24HC in human physiology and disease is being increasingly recognized, and because the current methods for identifying 24HC are time consuming and require expensive equipment, there is a need for a simpler assay for 24HC. The present invention addresses that need.